ABSTRACT Understanding the molecular drivers of biologically aggressive behavior in breast cancer will improve diagnosis and treatment. We have discovered that EZH2 overexpression in breast carcinomas is associated with ER negative (ER-) status, and is an independent biomarker of poor survival. While the oncogenic role of EZH2 in breast and other malignancies has been established, the mechanisms are far from clear. EZH2 is the histone H3 lysine 27 methyltransferase of the Polycomb Repressive Complex 2 (PRC2), responsible for epigenetic silencing. However, recent studies show that in invasive carcinomas with poor outcome, high EZH2 is associated with low H3K27me3, suggesting that in a subset of aggressive tumors, EZH2 functions through non-canonical H3K27me3-independent mechanisms. In the previous cycle, we have reported one such mechanism by which EZH2 activates NOTCH1. We have now discovered a new mechanism by which p38?-mediated phosphorylation of EZH2 at T367 leads to EZH2 cytoplasmic localization to promote migration and invasion of breast cancer cells. Using breast cancer tissues, our new preliminary data show that cytoplasmic pEZH2 T367 is significantly upregulated in distant metastasis compared to their matched primary tumors, and that primary tumors with high pEZH2 T367 metastasize earlier than those with low pEZH2. Previous studies have identified EZH2 in the cytoplasm, but the significance to human cancer was unknown. Further, p38? has been reported to phosphorylate EZH2 at T367 in muscle stem cells, but this event has never been linked to cytoplasmic EZH2 or with cancer. Our central hypothesis is that phosphorylated EZH2 at T367 promotes biologically aggressive breast cancer by localizing to the cytoplasm to enhance tumor cell migration, invasion and metastasis through H3K27me3-independent methyltransferase activity. We propose four independent hypothesis-driven aims. AIM 1: To investigate the consequences of inducible mammary- specific EZH2 overexpression on breast cancer progression, and the in vivo relevance of EZH2 non- canonical mechanisms. AIM 2: To elucidate the effect of pEZH2 T367 on neoplastic functions in vivo and in vitro. AIM 3: To investigate the molecular mechanism of pEZH2 T367-mediated breast cancer progression. AIM 4: To evaluate the translational impact of EZH2 non-canonical pathways in human breast tissue samples. We have developed critical reagents including an anti-pEZH2(T367) antibody, EZH2 T367A and T367D mutants (phospho-OFF and phospho-ON, respectively), characterized unique cohorts of human breast cancer tissues (n=500) with well-annotated clinical information and over 10 years of follow-up, and have generated new critical preliminary data. Our innovative studies have the potential to discover novel biomarkers and lead to mechanism-based therapies against breast cancer with EZH2 non-canonical pathway activation.